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| United States Patent |
5,236,592
|
|
Dejardin
, et al.
|
August 17, 1993
|
Haemocompatible composite material and method of using thereof
Abstract
A composite material is disclosed that comprises a support membrane coated
with an additive that reduces the thrombogenic character of the support.
The support membrane is a copolymer of acrylonitrile and at least one
ionic or ionizable monomer. The additive is a copolymer of acrylonitrile
in which at least one of the comonomers is polyoxyethylene. The composite
material of the invention is useful in the treatment of blood, and in
particular, dialysis and plasmapheresis.
| Inventors:
|
Dejardin; Philippe (Strasbourg, FR);
Yan; Feng (Strasbourg, FR);
Schmitt; Adrien (Mundolsheim, FR)
|
| Assignee:
|
Hospal Industrie (Meyzieu, FR)
|
| Appl. No.:
|
719245 |
| Filed:
|
June 21, 1991 |
Foreign Application Priority Data
| Current U.S. Class: |
210/646; 210/500.35; 210/500.43; 424/487; 523/112 |
| Intern'l Class: |
B01D 061/00 |
| Field of Search: |
210/500.35,500.43,638,490,654,646
427/245,246
424/78,79,486,487,78.08,78.31,78.35
523/112
|
References Cited
U.S. Patent Documents
| Re27401 | Jun., 1972 | Wichterle | 210/500.
|
| 3616930 | Feb., 1971 | Muir | 210/500.
|
| 3799355 | Mar., 1974 | Salyer et al. | 210/500.
|
| 3839200 | Oct., 1974 | Gigou et al. | 210/500.
|
| 4025439 | May., 1977 | Kamada et al. | 210/500.
|
| 4056467 | Nov., 1977 | Christen et al. | 210/638.
|
| 4545910 | Oct., 1985 | Marze | 210/500.
|
| 4548769 | Oct., 1985 | Shimomura et al. | 210/500.
|
| 5071973 | Dec., 1991 | Keller et al. | 424/486.
|
| Foreign Patent Documents |
| 1327990 | Aug., 1973 | GB.
| |
Other References
Miyama et al., Journal of Applied Polymer Science, vol. 35, pp. 115-125
(1988).
|
Primary Examiner: Dawson; Robert A.
Assistant Examiner: Kim; Sun Uk
Attorney, Agent or Firm: Bell, Seltzer, Park & Gibson
Claims
We claim:
1. A composite material comprising a support membrane formed from a
copolymer of acrylonitrile and at least one ionic or ionizable monomer, on
which membrane is adsorbed an additive that reduces the thrombogenic
character of the support, said additive comprising:
a copolymer in which at least one of the monomers comprises a
polyoxyethylene unit with a degree of polymerization of at least three;
and in which
acrylonitrile is a comonomer.
2. The material of claim 1, wherein one of the comonomers of the additive
comprising the polyoxyethylene unit is methoxypolyethylene glycol
methacrylate.
3. The material of claim 1, wherein the degree of polymerization of the
polyoxyethylene is 23.
4. A composite material comprising a support membrane formed from a
copolymer of acrylonitrile and at least one ionic or ionizable monomer, on
which membrane is adsorbed an additive that reduces the thrombogenic
character of the support, said additive comprising a copolymer:
a) in which at least one of the monomers comprises a polyoxyethylene unit
with a degree of polymerization of at least three;
b) in which acrylonitrile is a comonomer; and
c) which additionally comprises a positively charged comonomer.
5. The material of claim 4, wherein the positively charged comonomer is a
quaternary ammonium.
6. The material of claim 4, wherein the positively charged comonomer is a
trimethylaminoethyl methacrylate halide.
7. The material of claim 4, wherein the additive consists essentially of:
a.) 23 mole % of methoxypolyethylene glycol methacrylate;
b.) 74.5 mole % of acrylonitrile; and
c.) 2.5 mole % of positively charged comonomer.
8. The material of claim 1 or 4, wherein the support membrane consists
essentially of a copolymer of acrylonitrile and sodium
methallylsulphonate.
9. A method of blood dialysis which comprises circulating blood into
contact with the composite material of claim 1 or 4.
Description
FIELD OF THE INVENTION
The present invention relates to composite materials useful in the
extracorporeal treatment of blood. More specifically, the materials of the
present invention may be used in dialysis and plasmapheresis. The
materials comprise a support membrane adsorptively coated with an additive
that reduces the thrombogenic character of the support.
BACKGROUND
Blood circulates through vessels of the human body and these vessels have a
surface adapted for this purpose, i.e., a nonthrombogenic surface.
Platelets do not adhere to such nonthrombogenic surfaces and so there is
no coagulation.
Extracorporeal circulation of blood for treatment purposes necessitates
contact between the blood and an artificial surface. Thus, in dialysis the
blood comes into contact with various hoses and the dialysis membrane. The
same applies to plasmapheresis, or exposure of the blood to immobilized
enzymes or other substances intended for purification.
When blood contacts an artificial surface, the coagulation system is
activated, depositing blood platelets, leading to thromboses, which occur
either locally in contact with the artificial surface, or more generally
within the human body.
The materials used for the extracorporeal circulation and treatment of
blood are selected for biocompatibility, and especially
haemocompatibility. However, this is not always sufficient. Therefore,
workers have attempted to improve such materials by means of various
treatments.
Thus, Miyama describes a material formed by graft copolymerization of
methoxypolyethylene glycol methacrylate and polyacrylonitrile thioamide.
Miyama et al., Journal of Applied Polymer Science, "Graft Copolymerization
of Methoxypoly(ethylene glycol) Methacrylate Onto Polyacrylonitrile and
Evaluation of Nonthrombogenicity of the Copolymer", 35, 115-125 (1988).
The authors disclosed an improvement in the nonthrombogenic character of
the graft copolymer compared to the acrylonitrile polymer.
However, obtaining such a copolymer necessitates the use of a particular
production process. Indeed, the techniques described by Miyama do not make
it possible to improve, using relatively simple operations, the
nonthrombogenic character of a support membrane without substantially
modifying its nature.
It is advantageous to be able to preserve the nature, as well as the
properties, of certain existing support membranes while at the same time
improving their haemocompatibility.
SUMMARY OF THE INVENTION
To this end, the present invention provides a composite material comprising
a support membrane formed from a copolymer of acrylonitrile and at least
one ionic or ionizable monomer, on which membrane is adsorbed an additive
that reduces the thrombogenic character of the support. The additive is a
copolymer in which at least one of the monomers is the polyoxyethylene
unit with a degree of polymerization greater than or equal to three, and
in which acrylonitrile is a comonomer.
The adsorption of the additive makes it possible to preserve the chemical
nature of the support membrane and, therefore, its properties. The
improvement in the adsorption of the additive on the support membrane is
achieved by virtue of the interactions existing between the acrylonitrile
groups of the additive and those of the support.
Thus, by virtue of a simple treatment process, it is possible to improve
markedly the nonthrombogenic character of a support.
By adsorption is meant physical bonds such as ionic bonds, Van der Waals
bonds, and hydrophilic and hydrophobic bonds.
Coating by adsorption is economical compared to other treatments such as
bulk grafting. Indeed, maximum efficacy is achieved for a minimum quantity
of additive used, since a superficial layer of additive is sufficient to
obtain the desired effect.
According to a particularly advantageous embodiment of the invention, the
degree of polymerization of the polyoxyethylene is equal to 23.
Thus, the polyoxyethylene chains are sufficiently long to cause, at the
surface of the membrane, a certain steric hindrance which prevents the
deposition of platelets and fibrinogen. In addition, the hydrophilic
character of the polyoxyethylene leads to the formation of an aqueous
environment at the surface of the support that contributes to the
biocompatible character of the membrane.
According to a particular embodiment of the present invention, the additive
additionally comprises a positively charged comonomer.
This positive charge, which can derive from a quaternary ammonium for
example, improves the anchoring between the additive and the support by
virtue of a ionic bond.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows, diagramatically and with no specific scale, an experimental
set-up which makes it possible to demonstrate the improvement in the
nonthrombogenetic properties of the material according to the invention.
FIG. II illustrates the reduction in the platelet adhesion by the material
of the invention compared to a material of the prior art, in the absence
of red blood cells.
FIG. III illustrates the reduction in the platelet adhesion induced by the
material according to the invention compared to a material of the prior
art, in the presence of red blood cells.
FIGS. IV, V and VI illustrate the assay results of the plasma levels of
fibrinopeptide A, Beta-thromboglobulin, and activated protein C3a,
respectively, in X-vivo dialysis tests using materials of the present
invention as compared to material of the prior art.
DETAILED DESCRIPTION
The support membranes according to the present invention consist of a
copolymer of acrylonitrile and at least one ionic or ionizable monomer,
such as those described in the publishied patent specification GB
1,327,990. The ionic or ionizable monomers are essentially monomers that
are olefinically unsaturated and comprise at least one functional group
such as the sulphonic acid group or phosphonic acid group. The acid groups
are preferably in the form of salts, such as the sodium, potassium or
ammonium salts.
A preferred example of a support membrane is a membrane of acrylonitrile
and sodium methallylsulphonate, such as the dialysis membrane HOSPAL AN
69.RTM.. A plasmapheresis membrane consisting of methyl methacrylate and
acrylonitrile is another example of a preferred membrane.
The additive of the present invention is a copolymer wherein one of the
comonomers is acrylonitrile and at least one of the comonomers is a
polyoxyethylene macromonomer (CH.sub.2 CH.sub.2 O).sub.n. The degree of
polymerization (n) of the polyoxyethylene macromonomer (macromer) is
variable, but greater than or equal to three. In fact, the greater the
degree of polymerization, the greater the length of the chains and,
therefore, the more the aqueous environment, which is created by the
hydrophilicity of the polyoxyethylene, will represent a thin layer. This
environment is extremely haemocompatible. In addition, the polyoxyethylene
chains cause a steric hindrance at the surface of the support, which
prevents the deposition of platelets and the adsorption of fibrinogen.
It has been suggested that these flexible chains act as cilia, whose
movement prevents the deposition of platelets or fibrinogen.
Among the polyoxyethylene macromonomers suited to the present invention,
particular mention is made of triethylene glycol polyacrylate,
nonaethylene glycol methacrylate, methoxynonaethylene glycol methacrylate
and methoxy-23-ethylene glycol methacrylate.
In order to improve the anchoring of the additive to the support, at least
one positively charged monomer is added to the additive. In the case of a
support having electronegative sites, such as the sulphonate sites of the
membrane HOSPAL AN 69 (a registered trademark of Hospal Industrie), this
positive charge will permit the creation of ionic bonds strengthening the
adsorption of the additive to the support. The positive charge can be that
of a quaternary ammonium, e.g., trimethylaminoethyl methacrylate chloride.
The adsorption of the additive on the support membrane can be effected by
slowly circulating the additive, in contact with the support membrane, in
a buffer solution at the suitable pH, then by rinsing the support
copiously with this buffer solution.
In the case where the support membrane is in the form of hollow fibers
arranged inside a haemodialyser, the adsorption can be effected by slow
injection of the additive into a Tyrode buffer (NaCl, KCl, NaHCO.sub.3,
NaH.sub.2 PO.sub.4 --H.sub.2 O) for example.
The qualities exhibited by the composite material according to the present
invention, i.e., reducing the adsorption of fibrinogen and the adhesion of
platelets, can be tested by measuring radioactivity after labelling the
fibrinogen and the platelets, for example with iodine-125 and indium-111,
respectively.
EXAMPLE 1
An additive suitable for the present invention is produced by radical
copolymerization of acrylonitrile and methoxypolyethylene glycol
monomethacrylate. A(n) designates the polyethylene glycol methacrylate,
whose degree of polymerization is n, and B designates the acrylonitrile.
Dimethylformamide (DMF), dimethyl sulphoxide (DMSO) and the acrylonitrile
(B) are distilled twice over CaHz.sub.2, at reduced pressure. The
azobisisobutyronitrile used as initiator is crystallized from a methanol
toluene solution.
The methoxypolyethylene glycol methacrylates A(5), A(8) and A(21) are used
directly as provided by Polysciences.
The monomers, the solvent, and the initiator are introduced into a
double-walled glass reaction vessel equipped with a magnetic or mechanical
stirrer and connected to an external thermostat allowing the temperature
to be controlled to .+-.0.1.degree. C.
The system is then degassed by means of 3 successive cycles of evacuating
and flushing with argon, and the reaction is then carried out at a
constant temperature of 60.degree. C. under a slight argon pressure.
The consumption of acrylonitrile is monitored by gas-liquid chromatography.
The recovery and purification of the copolymers are effected using one of
the following procedures, depending on the composition of the sample:
a) the copolymers low in polyoxyethylene methacrylate are precipitated from
the reaction medium in distilled water in excess (10-fold), and they are
then purified by precipitation in water from their solution in DMF,
followed by rough washing with methanol in a mixer; or
b) the copolymers rich in polyoxyethylene methacrylate are precipitated
from the reaction medium in diethyl ether in excess (ten-fold); they are
then dissolved in distilled water, and the solutions are then completely
dialyzed for at least 48 hours, using Spectrapor cellulose membranes whose
cutoff threshold is 3,000 daltons.
The copolymers are then lyophilized if it is desired to preserve them.
EXAMPLE 2
An additive suitable for the present invention is produced by
copolymerization of the following reaction mixture:
commercial trimethylaminoethyl methacrylate chloride,
acrylonitrile,
methoxypolyethylene glycol methacrylate whose degree of polymerization is
23. The copolymerization is carried out at 30.degree. C.,
in the presence of KClO.sub.3 and NaHSO.sub.3 in aqueous medium.
The table below shows, for two samples X and Y, the molar composition in
monomers in the initial reaction mixture (f) and in the copolymer obtained
(F), as well as the corresponding mass composition (W) in the copolymer.
TABLE 1
______________________________________
SAMPLE A B C
______________________________________
X f 0.025 0.028
0.947
Not soluble F 0.019 0.187
0.794
in water W 0.015 0.810
0.165
Y f 0.033 0.050
0.917
Soluble F 0.026 0.228
0.746
in water W 0.018 0.840
0.132
______________________________________
Monomer A: Trimethylaminoethyl methacrylate chloride
Monomer B: Polyethylene glycol macromer (MG 23)
Monomer C: Acrylonitrile
EXAMPLE 3
Fibers consisting of a copolymer of acrylonitrile and sodium
methallylsulphonate are used as the support membrane: HOSPAL AN 69.TM..
These fibers, which are 48 in number, are inserted in a haemodialysis
mini-module. The internal diameter of the fibers is 270 .mu.m and their
length is approximately 20 cm.
The additive used is the terpolymer Y obtained in Example 2. This
terpolymer Y is in solution in ST (Simple Tyrode) buffer whose composition
1 liter is as follows:
NaCl: 8 g
KCl: 0.2 g
NaHCO.sub.3 : 1 g
NaH.sub.2 PO.sub.4 -H.sub.2 O: 0.06 g
In order to obtain the composite material of the present invention, the
internal surface of the hollow fibers is treated by adsorption, by slowly
injecting inside the mini-module a solution of terpolymer Y in the ST
buffer, and by then rinsing copiously with this buffer, whose pH is 7.5.
The parameters of this adsorption are the following:
concentration of the terpolymer in solution: 5 g/l
volume of solution injected: 25 ml
rate of injection: 0.1 ml/min
ambient temperature
EXAMPLE 4
The adsorption of fibrinogen is tested on a mini-module obtained in
accordance with Example 3.
To this end, a solution of fibrinogen is labelled with iodine-125 in
accordance with the Iodo-gen technique.
The fibrinogen adsorption kinetics are effected using the experimental
set-up described below:
Two syringe pushers P1 and P2, one containing the solution and the other
containing the solvent, are controlled by a microcomputer M1. The total
output, the concentration of the injected solution, the duration and the
number of passages of the solution can thus be programmed. Connected via a
Teflon tube to the two syringe pushers, the mini-module M is placed in a
slot in a detector (NaI crystal), which is connected to a counting scale
Ech, which is also linked to the microcomputer M1. On leaving the module
M, the solution is first directed towards a UV cell for checking the
protein concentration; it is then collected in a vessel positioned on
electronic scales BL, of which the data is acquired by the microcomputer
M1. All the instantaneous kinetic data are displayed on the screen and
recorded in a register, which can be used for a subsequent treatment.
The thermostating of the mini-module at 37.degree. C. is effected by
circulating the buffer in the dialysate compartment. The flow rate of this
buffer is controlled by a flowmeter.
The fibrinogen solution is introduced into the mini-modules by means of the
displacement of the buffer, in order to prevent the formation of an
air-solution interface. The passage of the radioactive suspension or
solution is framed by two passages of buffer of the same duration.
The duration of the passage of the solution and the duration of each
rinsing by means of the buffer are equal to 25 min. The flow rate is fixed
at 2 ml/min.
Following experiment, the fiber bundles are cut into 2-cm pieces in order
to determine the surface concentration as a function of the position on
the bundle.
In order to show the efficacy of the additive as regards to the reduction
in the adsorption of the fibrinogen, the same experiment is carried out on
a mini-module of hollow fibers consisting of the composite material
according to the present invention (obtained in accordance with Example 3)
and a mini-module of hollow fibers consisting of the HOSPAL AN
69.TM.membrane.
The table below collates the results obtained for 3 series of comparative
tests, carried out using solutions of different fibrinogen concentrations.
TABLE 2
______________________________________
Fibrinogen k
concentra- .gamma.
Slope (10.sup.-5
.GAMMA.
Module
tion (g/l) (S.sup.-1)
(CPM/s)
cm/s) (g/cm.sup.2)
R
______________________________________
A 0.0055 370 159.5 101.5 0.67 0
A 0.0063 370 17.3 11.0 0.085 88.0
treated
B 0.0050 518 139 105.5 0.73 0
B 0.0047 522 7.36 4.0 0.019 97.0
treated
C 0.339 505 563 9.89 4.86 0
C10 0.316 505 133 3.07 0.618 87.3
treated
______________________________________
.gamma. expresses the rate of shearing.
.GAMMA. represents the superficial interface concentration of fibrinogen
at a time t.
k is the apparent adsorption constant of the fibrinogen, which permits
comparison of the results, without the fibrinogen concentration of the
solution.
The slope, expressed in CPM/s (counts per minute per second) is
representative of the kinetics of the adsorption phenomenon.
In these three series of tests it is observed that the mini-modules
containing a material according to the invention adsorb for less
fibrinogen than do the controls, the reduction varying between 87 and 97%.
EXAMPLE 5
The kinetics of platelet adhesion are studied comparatively, using a
mini-module of hollow fibers consisting of composite material according to
the present invention, and with a mini-module of hollow fibers of HOSPAL
AN 69.TM. membrane serving as a control The experimental set-up is that of
the preceding example.
The platelets are labelled with indium-111 in TA buffer (Tyrode Albumin
0.35%). The duration for the passage of the platelet suspension is
variable (5 or 10 minutes); in contrast, the time for each rinsing is
fixed at 3 minutes.
Our results show an nonthrombogenic character of the material according to
the invention compared to the control-membrane
Our results were plotted as the surface platelet concentration as a
function of the distance with respect to the entry of the mini-module. The
platelet concentration of the solution used is 300,000 pl/.mu.1, the
duration of contact between the surface of the fibers and the platelet
solution is 10 minutes; the rate of shearing is 500 S.sup.-1, and the
reaction is carried out at 37.degree. C. The platelet solution contains no
red blood cells.
In the absence of red blood cells, the platelet adhesion is low, but the
composite material according to the invention produces a platelet adhesion
which is 40 to 50% lower than the platelet adhesion observed on the
control fibers.
EXAMPLE 6
The experimental conditions are the same as those in the preceding example.
The platelets are dissolved in a solution containing 40% of red blood
cells, which correspond fairly well to the concentration of the blood in
the case of the normal haematocrit. The platelet concentration is thus
180,000 pl/.mu.1.
The rate of shearing is 500 S.sup.-1 and the reaction is carried out at
ambient temperature.
Our results were charted for each of the mini-modules (according to the
invention and according to the control module), by plotting activity as a
function of time.
Under the conditions, once again the platelet adhesion is distinctly less
when using the composite material according to the invention than when
using the control hollow fibers.
EXAMPLE 7
The biocompatibility of the material of the subject invention is evaluated
by measuring the levels of fibrinopeptide A,.beta. thromboglobulin and C3
a protein during ex-vivo dialysis sessions.
To this end, use is made of mini-dialysers with hollow fibers whose
characteristics are as follows;
HOSPAL AN 69.TM. membrane;
internal diameter of fibers: 240 .mu.m;
external diameter of fibers: 340 .mu.m;
number of fibers: 340;
inner surface: 512 cm.sup.2 ;
After rinsing the mini-dialysers with an aqueous solution of NaCl at 0.9
g/l, two of them are treated by adsorption of the additive Y obtained in
accordance with Example 2. A third one is conserved intact to serve as a
control. The treatment by adsorption is carried out by passing through the
fibers a polymer solution at 5 g/l in a simple tyrode buffer.
5 ml are passed at a rate of 6 ml/min, then 17 ml at a rate of 0.1 ml/min.
After treatment, the dialysers are rinsed using an aqueous solution of NaCl
at 0.9 g/l, heparinized at 2.5 IU/ml for the blood compartment.
To carry out the ex-vivo tests, the rinsing liquid is retained in the
dialysate compartment by closing the intake and outlet points of the
dialysis fluid, and the blood of a donor is passed into the blood
compartment at a rate of 10 ml/min for 34 min. The rate of shearing is 360
S.sup.-1. After its passage through the haemodialyser, the blood is not
returned to the patient.
The assay of a certain number of plasmatic substances is then carried out,
which makes it possible to evaluate the biocompatability of the material.
We determined the plasma level of fibrinopeptide A in two tests carried out
with the material of the invention, as well as the control consisting of a
HOSPAL AN 69.TM. membrane without additive.
Fibrinopeptide A is an early indicator of activation of the phenomenon of
coagulation.
Our results show a clear improvement in the nonthrombogenic character of
the material of the present invention.
We also determined the plasma levels of .beta.-thromboglobulin, which is an
indicator of the platelet activation.
Here again, we observed the highly satisfactory performance of the material
of the present invention compared to the prior art.
We also performed a plasma assay of activated protein C3a, the presence of
which indicates activation of the complement system.
The results obtained with the material of the invention are substantially
equal to those obtained with the HOSPAL AN 69.TM. membrane which is
already renowned for its performance in this regard. Thus, the high
quality of the support is maintained upon adsorption of the additive Y.
EXAMPLE 8
The sustaining of the performance (dialysis and permeability) of the
material of the invention compared to the support not covered with the
additive is verified.
To this end, use is made of a mini-dialyzer equipped with 170 fibers of
HOSPAL AN 69.RTM. membrane.
The following measurements are effected on this dialyzer:
Ultrafiltration
Urea clearance
Vitamin B.sub.12 clearance.
After carrying out these measurements, the dialyzer is treated by
adsorption of the terpolymer Y in the same way as in the preceding
example. A material according to the present invention is thus obtained.
The ultrafiltration is once again measured, as are the urea clearance and
vitamin B.sub.12 clearance.
The measurements carried out are corrected to bring them to the same flow
rate conditions at the inlet of the blood compartment (namely 3.5 ml/min)
and of the dialysate compartment (namely 10.5 ml/min), the ultrafiltration
flow rate being zero.
The results before and after treatment are collected in the table below:
TABLE 3
______________________________________
Ultrafiltration
Urea
Clearance in ml/H .multidot. m.sup.2 .multidot. mmHg
Clearance Vit B.sub.12
______________________________________
Before 52 3.1 1.5
treatment
After 48 3.1 1.5
treatment =
invention
______________________________________
The results show that the performance of the material are sustained when it
is covered by adsorption with the additive Y. (The apparent fall as
regards ultrafiltration is no doubt due to the conditions of the
measurement, which it was only possible to carry out after 2 days).
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